CN112424596A - 生物膜磷酸肌醇的分离方法 - Google Patents
生物膜磷酸肌醇的分离方法 Download PDFInfo
- Publication number
- CN112424596A CN112424596A CN201980046921.2A CN201980046921A CN112424596A CN 112424596 A CN112424596 A CN 112424596A CN 201980046921 A CN201980046921 A CN 201980046921A CN 112424596 A CN112424596 A CN 112424596A
- Authority
- CN
- China
- Prior art keywords
- separation
- pips
- separating
- biofilm
- isomers
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims description 15
- 150000003906 phosphoinositides Chemical class 0.000 title claims description 6
- 239000012528 membrane Substances 0.000 title claims description 4
- 238000000926 separation method Methods 0.000 claims abstract description 50
- 238000004808 supercritical fluid chromatography Methods 0.000 claims abstract description 13
- 229920000858 Cyclodextrin Polymers 0.000 claims abstract description 11
- 238000004458 analytical method Methods 0.000 claims abstract description 9
- 239000001116 FEMA 4028 Substances 0.000 claims abstract description 8
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims abstract description 8
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims abstract description 8
- 229960004853 betadex Drugs 0.000 claims abstract description 8
- 239000012530 fluid Substances 0.000 claims abstract description 7
- UZMPYXSDDZXMAI-OHKKONBVSA-N [(2r)-2-hexadecanoyloxy-3-[hydroxy-[(2r,3r,5s,6r)-2,4,6-trihydroxy-3,5-diphosphonooxycyclohexyl]oxyphosphoryl]oxypropyl] hexadecanoate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@@H](OC(=O)CCCCCCCCCCCCCCC)COP(O)(=O)OC1[C@H](O)[C@@H](OP(O)(O)=O)C(O)[C@@H](OP(O)(O)=O)[C@H]1O UZMPYXSDDZXMAI-OHKKONBVSA-N 0.000 claims description 8
- 239000003607 modifier Substances 0.000 claims description 8
- 238000001212 derivatisation Methods 0.000 claims description 7
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 7
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 6
- 229960000367 inositol Drugs 0.000 claims description 6
- -1 inositol phosphates Chemical class 0.000 claims description 6
- TZIHFWKZFHZASV-UHFFFAOYSA-N methyl formate Chemical compound COC=O TZIHFWKZFHZASV-UHFFFAOYSA-N 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 5
- 235000021317 phosphate Nutrition 0.000 claims description 5
- 150000003915 phosphatidylinositol 4,5-bisphosphates Chemical class 0.000 claims description 4
- ONDSBJMLAHVLMI-UHFFFAOYSA-N trimethylsilyldiazomethane Chemical compound C[Si](C)(C)[CH-][N+]#N ONDSBJMLAHVLMI-UHFFFAOYSA-N 0.000 claims description 4
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 claims description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 3
- 238000002370 liquid polymer infiltration Methods 0.000 abstract description 36
- 239000000523 sample Substances 0.000 description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 6
- 150000001982 diacylglycerols Chemical class 0.000 description 6
- 239000012488 sample solution Substances 0.000 description 6
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000001569 carbon dioxide Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical group O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 150000003905 phosphatidylinositols Chemical class 0.000 description 3
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 3
- 239000000377 silicon dioxide Substances 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000020176 deacylation Effects 0.000 description 2
- 238000005947 deacylation reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- QZHDNICXKYAEKK-UHFFFAOYSA-N diazomethyl(trimethyl)silane;hexane Chemical compound CCCCCC.C[Si](C)(C)C=[N+]=[N-] QZHDNICXKYAEKK-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000012299 nitrogen atmosphere Substances 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- SVWLIIFHXFGESG-UHFFFAOYSA-N formic acid;methanol Chemical compound OC.OC=O SVWLIIFHXFGESG-UHFFFAOYSA-N 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3204—Inorganic carriers, supports or substrates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/40—Selective adsorption, e.g. chromatography characterised by the separation mechanism using supercritical fluid as mobile phase or eluent
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/282—Porous sorbents
- B01J20/285—Porous sorbents based on polymers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/16—Injection
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Inorganic Chemistry (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
本发明提供一种能够在不将PIPs进行脱酰基化的情况下分离PIPs的异构体的分离方法。此分离方法至少包括分离步骤，在包括分离管柱的超临界流体色谱仪的分析流路中注入包含多种PIPs的试样，所述分离管柱在内部填充有包含β‑环糊精的分离介质，且利用超临界流体色谱法来将所述多种PIPs相互分离。
Description
技术领域
本发明涉及一种生物膜磷酸肌醇的分离方法，所述生物膜磷酸肌醇是磷脂酰肌醇(phosphatidylinositol)的肌醇(inositol)环的3,4,5位羟基受到磷酸化的磷脂质。
背景技术
如图1所示，磷酸肌醇(phosphoinositide)(以下，PIPs)中，存在受到磷酸化的数量或位置彼此不同的PI(3)P、PI(4)P、PI(5)P、PI(3,4)P2、PI(3,5)P2、PI(4,5)P2、PI(3,4,5)P3这7种。
在二酰基甘油(diacyl glycerol，DG)相同的情况下，PI(3)P、PI(4)P、PI(5)P这3种、PI(3,4)P2、PI(3,5)P2、PI(4,5)P2这3种分别为异构体且具有相同的质量，因此为了将7种PIPs各别地定量，而需要利用色谱法来分离所述异构体。
然而，利用色谱法来分离PIPs的异构体的方法并未确立。因此，迄今为止，采用不将PIPs的异构体分离而一起定量的方法、或将PIPs的二酰基甘油切断(脱酰基化)而使用肌醇环部分来分离异构体的方法(参照非专利文献1、非专利文献2)。
[现有技术文献]
[非专利文献]
非专利文献1：《使用质谱仪的肌醇磷脂质的一齐定量分析法的开发》，中西广树、佐佐木纯子、佐佐木雄彦等人，脂质生物化学研究，第54卷第88页～第89页，2012年
非专利文献2：《用于微量脂质成分测定的质谱技术的现状》，田口良、中西广树，实验医学第28卷第20期(增刊)，2010年
发明内容
[发明所要解决的问题]
不将PIPs的异构体分离而一起定量的方法中，将PI(3)P、PI(4)P、PI(5)P设为PIP1，将PI(3,4)P2、PI(3,5)P2、PI(4,5)P2设为PIP2，对二酰基甘油(DG)的不同种类进行定量。因此，无法对每种异构体进行定量。
另外，将PIPs进行脱酰基化而使用肌醇环部分来分离异构体的方法中，能够对每个肌醇环部分进行定量，另一方面，无法获得与DG相关的信息。
本发明的目的在于提供一种能够在不将PIPs进行脱酰基化的情况下分离PIPs的异构体的分离方法。
[解决问题的技术手段]
本发明者们发现，通过超临界流体色谱法，能够在不进行脱酰基化的情况下分离PIPs的异构体，所述超临界流体色谱法使用如下分离管柱，即，填充有在载体上结合有β-环糊精的分离介质的分离管柱。认为其原因在于，在分子形状识别能力比液相色谱法高的超临界色谱法中，通过使用包含β-环糊精的介质来作为分离介质，而如图4所示，对PIPs发挥疏水性相互作用、氢键合、包合、静电性相互作用等多种作用，迄今为止难以分离的非脱酰基化状态的PIPs的异构体相互分离。此外，图4的R1是包含烷基及极性基的间隔物。
即，本发明的PIPs的分离方法包括分离步骤，在包括分离管柱的超临界流体色谱仪的分析流路中注入包含多种PIPs的试样，所述分离管柱在内部填充有包含β-环糊精的分离介质，且利用超临界流体色谱法来将所述多种PIPs相互分离。
因此，本发明的分离方法适合于包含PIPs的多种异构体的试样的分离。
所谓所述多种异构体，是指PI(3)P、PI(4)P、PI(5)P、PI(3,4)P2、PI(3,5)P2、PI(4,5)P2、PI(3,4,5)P3中的任一者。
优选为在所述分离步骤之前包括衍生化步骤，利用三甲基硅烷基-重氮甲烷，将试样中所含的所述多种PIPs的磷酸基进行衍生化；且在所述分离步骤之后包括检测步骤，利用质谱仪来分别检测由所述分离管柱所分离的所述多种PIPs。如此一来，能够利用质谱仪，对包含经过分离管柱而分离的异构体的各PIPs进行定量分析，所述分离管柱在内部填充有包含β-环糊精的分离介质，因此能够实现试样中所含的多种PIPs的各别定量。
在所述分离步骤中，能够将甲酸甲醇水溶液用作改性剂。
[发明的效果]
本发明的PIPs的分离方法包括如下分离步骤，即，在包括分离管柱的超临界流体色谱仪的分析流路中注入包含多种PIPs的试样，所述分离管柱在内部填充有包含β-环糊精的分离介质，且利用超临界流体色谱法来将所述多种PIPs相互分离，故而能够在不进行脱酰基化的情况下分离PIPs的异构体。
附图说明
图1是表示磷脂酰肌醇及各PIPs的结构的结构式。
图2是表示超临界流体色谱仪的结构的流路结构图。
图3是表示PIPs的分离方法的一实施例的流程图。
图4是用以对包含β-环糊精的分离介质与PIPs之间的相互作用加以说明的图。
图5是以利用所述实施例的分离方法而获得的质谱仪的信号为基础的色谱图的一例。
具体实施方式
以下，参照图式，对本发明的PIPs的分离方法的一实施例加以说明。
所述实施例的分离方法是使用超临界流体色谱仪(Supercritical FluidChromatograph)(以下，SFC)来实施。此实施例中使用的SFC如图2所示，包括：送液泵4、送液泵6，用以在分析流路2中输送二氧化碳与改性剂；试样注入部8，用以在二氧化碳与改性剂的混合流体所流通的分析流路2中注入试样；分离管柱10，用以将通过试样注入部8而注入的试样进行分离；背压控制器(Back Pressure Regulator，BPR)12，以至少在分离管柱10中流通的二氧化碳成为超临界状态的方式，将分析流路2内的压力控制为预定压力；泵15，输送用以灵敏度良好地进行检测的补给；以及质谱仪(Mass Spectrometry，MS)14，设置于BPR12的后段侧。
虽省略图示，但分离管柱10收纳于管柱烘箱内，固定地控制为所设定的温度。分离管柱10填充有能够包合有机物的环糊精结合于二氧化硅载体上的分离介质，例如，能够使用信和化工股份有限公司制造的奥创(ULTRON)AF-HILIC-CD。
为了能够利用所述SFC来对包含多种PIPs的试样进行分离分析，而将试样中的PIPs的磷酸基进行衍生化，成为能够利用MS 14来检测各PIPs的状态。
衍生化的处理例如能够通过以下(1)～(5)的次序来进行。
(1)在包含PIPs的试样溶液中添加2M的三甲基硅烷基-重氮甲烷己烷溶液。
(2)将添加有2M的三甲基硅烷基-重氮甲烷己烷溶液的试样溶液在室温下放置一定时间(例如10分钟)，进行衍生化反应。
(3)在氮气环境下向试样溶液中添加冰乙酸，使衍生化反应停止。
(4)将预定的洗涤液(例如氯仿：甲醇：水＝8：4：3的混合液)添加至试样溶液中并混合后，进行离心分离而回收下层。也可将同样的洗涤反复进行多次。最后在试样溶液中添加甲醇：水＝9：1的溶液。
(5)在氮气环境下使试样溶液干燥凝固。然后，在试样中添加预定量的甲醇，利用超声波使其溶解。进而将预定量的水添加至试样中。
如上所述的PIPs的衍生化公开于论文“《利用质谱法的细胞与组织中的PtdInsP3分子种类的定量(Quantification of PtdInsP3 molecular species in cells andtissues by mass spectrometry)》，乔纳森·克拉克(Jonathan CLark)、凯伦·E·安德森(Karen E Anderson)、维罗妮克·朱文(Veronique Juvin)、特雷弗·S·史密斯(Trevor SSmith)、弗雷德里克·卡普(Fredrik Karpe)、迈克尔·J·O·韦克兰(Michael J OWakelam)、兰·R·史蒂芬斯(Len R Stephens)与菲利普·T·霍金斯(Phillip THawkins)”。
超临界流体色谱法的改性剂能够使用0.1％甲酸甲醇与水的混合液(例如甲酸甲醇：水＝97.5：2.5)。另外，能够使用包含甲酸或甲酸铵的甲醇(例如0.1％甲酸甲醇)来作为改性剂。
即，所述实施例的PIPs的分离方法如图3的流程图所示，在实施所述的衍生化处理之后(步骤S1)，通过试样注入部8将试样注入至SFC的分析流路2中(步骤S2)，利用填充有环糊精结合于二氧化硅载体上的分离介质的分离管柱10来分离PIPs的异构体(步骤S3)。进而，将在分离管柱10中分离的各PIPs依序导入至MS 14来进行检测(步骤S4)。
图5是将包含PI(3)P、PI(4)P、PI(5)P、PI(3,4)P2、PI(3,5)P2、PI(4,5)P2、PI(3,4,5)P3这7种PIPs的试样，使用所述实施例的方法进行分析而获得的色谱图，横轴为时间，纵轴为信号强度。在所述分析中，使用信和化工股份有限公司制造的奥创(ULTRON)AF-HILIC-CD(内径为4.6mm，长度为250mm)作为分离管柱10，且将分离管柱10的设定温度设为4℃。另外，使用0.1％甲酸甲醇与水的混合液(例如甲酸甲醇：水＝97.5：2.5)来作为改性剂，使流动相中的改性剂浓度在0分钟～7分钟的时间带中变化为5％，在7.01分钟～10分钟的时间带中变化为30％，在10.01分钟～16分钟的时间带中变化为5％。将流动相的流量设为3mL/min，将补给的流量设为0.1mL/min，且将BPR 12的设定压力设为10MPa。
根据图5的色谱图可知，彼此为异构体的PI(3)P、PI(4)P、PI(5)P这3种、PI(3,4)P2、PI(3,5)P2、PI(4,5)P2这3种分别分离。PI(3)P、PI(4)P、PI(5)P这3种、PI(3,4)P2、PI(3,5)P2、PI(4,5)P2这3种分别具有相同的质量，但由于利用SFC来分离，故而能够利用MS 14来各别地检测各异构体并定量。
根据以上可知，能够利用包括分离管柱的SFC与MS的组合来进行7种PIPs的各别定量，所述分离管柱填充有环糊精结合于二氧化硅载体上的分离介质。
[符号的说明]
2：分析流路
4、6、15：送液泵
8：试样注入部
10：分离管柱
12：背压控制器(BPR)
14：质谱仪(MS)。
Claims (5)
1.一种生物膜磷酸肌醇的分离方法，包括分离步骤：在包括分离管柱的超临界流体色谱仪的分析流路中注入包含多种生物膜磷酸肌醇的试样，所述分离管柱在内部填充有包含β-环糊精的分离介质，且利用超临界流体色谱法来将所述多种生物膜磷酸肌醇相互分离。
2.根据权利要求1所述的分离方法，其中所述多种生物膜磷酸肌醇包含生物膜磷酸肌醇的多种异构体。
3.根据权利要求2所述的分离方法，其中所述多种异构体为PI(3)P、PI(4)P、PI(5)P、PI(3,4)P2、PI(3,5)P2、PI(4,5)P2、PI(3,4,5)P3中的任一者。
4.根据权利要求1所述的分离方法，其中在所述分离步骤之前包括衍生化步骤，利用三甲基硅烷基-重氮甲烷，将所述多种生物膜磷酸肌醇的磷酸基进行衍生化；并且
在所述分离步骤之后包括检测步骤：利用质谱仪，来分别检测由所述分离管柱所分离的所述多种生物膜磷酸肌醇。
5.根据权利要求1所述的分离方法，其中在所述分离步骤中，将甲酸甲醇水溶液用作改性剂。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018168584 | 2018-09-10 | ||
| JP2018-168584 | 2018-09-10 | ||
| PCT/JP2019/034134 WO2020054462A1 (ja) | 2018-09-10 | 2019-08-30 | 生体膜ホスホイノシタイドの分離方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112424596A true CN112424596A (zh) | 2021-02-26 |
| CN112424596B CN112424596B (zh) | 2023-07-18 |
Family
ID=69778299
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201980046921.2A Active CN112424596B (zh) | 2018-09-10 | 2019-08-30 | 生物膜磷酸肌醇的分离方法 |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US12117426B2 (zh) |
| JP (1) | JP7017704B2 (zh) |
| CN (1) | CN112424596B (zh) |
| AU (1) | AU2019340961B2 (zh) |
| WO (1) | WO2020054462A1 (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102178630B1 (ko) * | 2018-12-20 | 2020-11-13 | 한화토탈 주식회사 | 프로필렌 중합용 고체 촉매 및 이를 이용한 블록 공중합체의 제조방법 |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2151397C1 (ru) * | 1999-11-22 | 2000-06-20 | Тверская государственная медицинская академия | Способ дифференциальной диагностики доброкачественной гиперплазии и рака предстательной железы |
| US20040018254A1 (en) * | 2002-07-24 | 2004-01-29 | Rabinowitz Israel N. | Synergistic compositions from yeast-modified aqueous extracts from almond hulls |
| US20040253656A1 (en) * | 2003-06-11 | 2004-12-16 | David Williams | Method for measuring inositol triphosphate |
| WO2012166916A1 (en) * | 2011-06-03 | 2012-12-06 | Waters Technologies Corporation | Method of separation of lipid and biological molecular species using high purity chromatographic materials |
| JP2015194363A (ja) * | 2014-03-31 | 2015-11-05 | 株式会社島津製作所 | 順相・逆相カラムを備えた超臨界流体クロマトグラフとそれを用いた分析方法 |
| JP2015215320A (ja) * | 2014-05-13 | 2015-12-03 | 株式会社島津製作所 | 異なる極性をもつ化合物を含む試料の一斉分析方法 |
| WO2016057924A1 (en) * | 2014-10-10 | 2016-04-14 | Genentech, Inc. | Pyrrolidine amide compounds as histone demethylase inhibitors |
| WO2017001658A1 (en) * | 2015-07-02 | 2017-01-05 | F. Hoffmann-La Roche Ag | Benzoxazepin oxazolidinone compounds and methods of use |
| WO2017147049A1 (en) * | 2016-02-22 | 2017-08-31 | The Research Foundation For The State University Of New York | Methods and compositions for substituting membrane lipids in living cells |
| WO2018016645A1 (ja) * | 2016-07-22 | 2018-01-25 | 国立大学法人秋田大学 | 新規リン脂質およびその利用ならびにリン脂質分離測定法の開発 |
| CN107703235A (zh) * | 2017-07-18 | 2018-02-16 | 广东研捷医药科技有限公司 | 一种来那度胺对映异构体超临界流体色谱分离方法 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0716362B2 (ja) * | 1990-09-26 | 1995-03-01 | 辻製油株式会社 | 高濃度のホスファチジルコリンを含むレシチンを採取する方法 |
| US20160266129A1 (en) * | 2015-03-11 | 2016-09-15 | Christopher Janetopoulos | Phosphoinositide (4,5) Bisphosphate as a Diagnostic Tool and Target for Cancer Treatment |
| CN105021758B (zh) * | 2015-07-27 | 2017-05-10 | 中国科学院生物物理研究所 | 一种基于化学衍生的磷脂分类检测和定量方法 |
-
2019
- 2019-08-30 JP JP2020545914A patent/JP7017704B2/ja active Active
- 2019-08-30 US US17/272,973 patent/US12117426B2/en active Active
- 2019-08-30 CN CN201980046921.2A patent/CN112424596B/zh active Active
- 2019-08-30 AU AU2019340961A patent/AU2019340961B2/en active Active
- 2019-08-30 WO PCT/JP2019/034134 patent/WO2020054462A1/ja active Application Filing
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2151397C1 (ru) * | 1999-11-22 | 2000-06-20 | Тверская государственная медицинская академия | Способ дифференциальной диагностики доброкачественной гиперплазии и рака предстательной железы |
| US20040018254A1 (en) * | 2002-07-24 | 2004-01-29 | Rabinowitz Israel N. | Synergistic compositions from yeast-modified aqueous extracts from almond hulls |
| US20040253656A1 (en) * | 2003-06-11 | 2004-12-16 | David Williams | Method for measuring inositol triphosphate |
| WO2012166916A1 (en) * | 2011-06-03 | 2012-12-06 | Waters Technologies Corporation | Method of separation of lipid and biological molecular species using high purity chromatographic materials |
| JP2015194363A (ja) * | 2014-03-31 | 2015-11-05 | 株式会社島津製作所 | 順相・逆相カラムを備えた超臨界流体クロマトグラフとそれを用いた分析方法 |
| JP2015215320A (ja) * | 2014-05-13 | 2015-12-03 | 株式会社島津製作所 | 異なる極性をもつ化合物を含む試料の一斉分析方法 |
| WO2016057924A1 (en) * | 2014-10-10 | 2016-04-14 | Genentech, Inc. | Pyrrolidine amide compounds as histone demethylase inhibitors |
| WO2017001658A1 (en) * | 2015-07-02 | 2017-01-05 | F. Hoffmann-La Roche Ag | Benzoxazepin oxazolidinone compounds and methods of use |
| WO2017147049A1 (en) * | 2016-02-22 | 2017-08-31 | The Research Foundation For The State University Of New York | Methods and compositions for substituting membrane lipids in living cells |
| WO2018016645A1 (ja) * | 2016-07-22 | 2018-01-25 | 国立大学法人秋田大学 | 新規リン脂質およびその利用ならびにリン脂質分離測定法の開発 |
| CN107703235A (zh) * | 2017-07-18 | 2018-02-16 | 广东研捷医药科技有限公司 | 一种来那度胺对映异构体超临界流体色谱分离方法 |
Non-Patent Citations (8)
| Title |
|---|
| JONATHAN CLARK 等: "Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry", 《NATURE METHODS》 * |
| JONATHAN CLARK 等: "Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry", 《NATURE METHODS》, vol. 08, no. 03, 31 March 2011 (2011-03-31), pages 267 - 272 * |
| PAVLINA T. IVANOVA 等: "Glycerophospholipid Identification and Quantitation by Electrospray Ionization Mass Spectrometry", 《METHODS IN ENZYMOLOGY》 * |
| PAVLINA T. IVANOVA 等: "Glycerophospholipid Identification and Quantitation by Electrospray Ionization Mass Spectrometry", 《METHODS IN ENZYMOLOGY》, vol. 432, 31 December 2007 (2007-12-31), pages 21 - 57 * |
| 王迎春 等: "超临界流体色谱法测定蛋黄卵磷脂中的8种磷脂组分", 《药物分析杂志》 * |
| 王迎春 等: "超临界流体色谱法测定蛋黄卵磷脂中的8种磷脂组分", 《药物分析杂志》, vol. 36, no. 02, 31 December 2016 (2016-12-31), pages 267 - 272 * |
| 邓启刚 等: "大豆肌醇磷脂的分离技术研究", 《化学工程师》 * |
| 邓启刚 等: "大豆肌醇磷脂的分离技术研究", 《化学工程师》, no. 06, 31 December 2003 (2003-12-31), pages 14 - 16 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2020054462A1 (ja) | 2020-03-19 |
| US20210310999A1 (en) | 2021-10-07 |
| AU2019340961A1 (en) | 2021-04-01 |
| JPWO2020054462A1 (ja) | 2021-08-30 |
| CN112424596B (zh) | 2023-07-18 |
| JP7017704B2 (ja) | 2022-02-09 |
| AU2019340961B2 (en) | 2022-07-14 |
| US12117426B2 (en) | 2024-10-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Schurig | Chiral separations using gas chromatography | |
| Wang et al. | Method development for the analysis of phthalate esters in tea beverages by ionic liquid hollow fibre liquid-phase microextraction and liquid chromatographic detection | |
| US20150068279A1 (en) | Electro-enhanced solid-phase microextraction method | |
| Stalikas et al. | Microextraction combined with derivatization | |
| CN110631875B (zh) | 一种选择性吸附并分步洗脱磷脂和糖鞘脂的方法 | |
| Frenkel et al. | Chiral separation of a diketopiperazine pheromone from marine diatoms using supercritical fluid chromatography | |
| CN112424596B (zh) | 生物膜磷酸肌醇的分离方法 | |
| Bian et al. | Progress in the pretreatment and analysis of N-nitrosamines: an update since 2010 | |
| CN102411034A (zh) | 直接衍生高效液相色谱法测定水基胶中羰基化合物的方法 | |
| Müller-Reif et al. | A new parallel high-pressure packing system enables rapid multiplexed production of capillary columns | |
| Poole et al. | Liquid Chromatography . | |
| CN106290663A (zh) | 一种烟用香料挥发性成分分析的前处理方法 | |
| CN105334282B (zh) | 一种地表水体中环境雌激素的共检测方法 | |
| CN103913525B (zh) | 一种高效液相色谱-串联质谱法检测白酒中17种塑化剂的方法 | |
| Fabel et al. | Effect-directed analysis by high-performance liquid chromatography with gas-segmented enzyme inhibition | |
| CN104458973A (zh) | 一种适用于玉米赤霉烯酮及其代谢物的在线测定方法 | |
| CN103721567A (zh) | 一种基于中空纤维纳滤膜材料的样品溶液净化装置 | |
| Hooijschuur et al. | In‐line sample trap columns with diatomite for large‐volume injection in CZE–IM–MS | |
| Lu et al. | Simultaneous analysis of endogenetic and ectogenic plant hormones by pressurized capillary electrochromatography | |
| Jiang et al. | Preparation of covalently bonded liposome capillary column and its application in evaluation of drug membrane permeability | |
| Boonjob et al. | In-line sequential injection-based hollow-fiber sorptive microextraction as a front-end to gas chromatography–mass spectrometry: a novel fully automatic sample processing technique for residue analysis | |
| CN102680590A (zh) | 用于检测水中四种胺类物的二维液相色谱-串联质谱方法 | |
| CN108845051B (zh) | 一种基于UHPLC-QTrap MS检测斑马鱼脑组织中鞘脂的方法 | |
| JP2008304435A (ja) | 多成分分析方法および多成分分析装置 | |
| Tu et al. | Identification and Characterization of Isomeric Intermediates in a Catalyst Formation Reaction by Means of Speciation Analysis Using HPLC− ICPMS and HPLC− ESI-MS |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |